Selective somatostatin receptor 5 inhibition improves hepatic insulin sensitivity

Abstract Diabetes is a metabolic disorder with an increasing global prevalence. Somatostatin (SST), a peptide hormone, regulates hormone secretion via five SST receptor (SSTR) subtypes (SSTR1–5) in a tissue‐specific manner. As SSTR5 is expressed in pancreatic β‐cells and intestinal L‐cells, studies have suggested that SSTR5 regulates glucose tolerance through insulin and incretin secretion, thereby having a prominent role in diabetes. Moreover, SSTR5 knockout (KO) mice display enhanced insulin sensitivity; however, the underlying mechanism has not been clarified. Therefore, in this study, we investigate the effect of SSTR5 blockade on insulin resistance and the target organ using SSTR5 KO mice and a selective SSTR5 antagonist (compound‐1). High‐fat diet (HFD)‐fed SSTR5 KO mice exhibited significantly lower homeostasis model assessment of insulin resistance (HOMA‐IR) than HFD‐fed wild‐type mice. Two‐week oral administration of compound‐1 dose‐dependently and significantly reduced changes in the levels of glycosylated hemoglobin (GHb), plasma glucose, plasma insulin, and HOMA‐IR in male KK‐Ay/Ta Jcl mice (KK‐Ay mice), a model of obese type 2 diabetes with severe insulin resistance. Additionally, compound‐1 significantly increased the glucose infusion rate while decreasing hepatic glucose production in male KK‐Ay mice, as evidenced by hyperinsulinemic‐euglycemic clamp analyses. In addition, compound‐1 ameliorated the insulin‐induced Akt phosphorylation suppression by octreotide in the liver of male C57BL/6J mice. Collectively, our results demonstrate that selective SSTR5 inhibition can improve insulin sensitivity by enhancing liver insulin action; thus, selective SSTR5 antagonists represent potentially novel therapeutic agents for type 2 diabetes.


| INTRODUC TI ON
The global prevalence of type 2 diabetes is steadily increasing. In 2021, the number of patients with diagnosed and undiagnosed type 2 diabetes among adults aged >20 years was approximately 537 million worldwide. By the year 2045, this number is estimated to reach 783 million, i.e., 1 in 10 adults will develop diabetes. Furthermore, according to the 10th edition of the International Diabetes Federation, in 2021, 541 million individuals had developed impaired glucose tolerance, a prediabetic condition; this number is projected to rise to 730 million by 2045. 1 Thus, type 2 diabetes remains a major global health issue.
Diabetes is a metabolic disease characterized by hyperglycemia due to insufficient insulin secretion, insulin inaction, or both. In diabetes, chronic hyperglycemia is associated with long-term damage and dysfunction of various organs, resulting in microvascular (e.g., nephropathy, retinopathy, and neuropathy) and macrovascular diseases (e.g., myocardial infarction, stroke, and peripheral arterial disease) associated with a high mortality rate. [2][3][4] The number of deaths attributable to diabetes was estimated to be 4.2 million in 2019, contributing to 11.3% of deaths worldwide. 5 Conversely, in a recent large clinical study, appropriate glycemic control by antidiabetic drugs significantly decreased the risk of developing micro-and macrovascular diseases, in particular cardiovascular events. [6][7][8][9] Insulin resistance is an initial indicator of abnormal glucose homeostasis 10,11 and is a strong risk factor for diabetic complications 12 ; thus, insulin sensitizers, including metformin, are used as the first-line treatment for type 2 diabetes. 13,14 Notably, a Diabetes Prevention Program study reported that metformin could effectively delay the onset of diabetes 15 and suppresses diabetes progression by preventing β-cell exhaustion and death caused by excessive insulin secretion.
Somatostatin (SST), a peptide hormone, was discovered in the hypothalamus as an inhibitor of growth hormone (GH) secretion 16 and has two biologically active forms, namely a 14 or 28 amino acid peptide hormone (SST14 and SST28, respectively) generated from preproSST (116 amino acids) in mammals. [16][17][18] SST14 is predominantly expressed in the pancreatic δ cells, stomach, and neural tissues, whereas SST28 is predominantly expressed in the mucosa of the ileum and colon. Both SST14 and SST28 bind to the SST receptor (SSTR), which belongs to the G-protein-coupled receptor family containing seven transmembrane domains. To date, five SSTR genes have been cloned (SSTR1-5) in humans and other species 19 ; SSTR2 is alternatively spliced to generate two isoforms, SSTR2A and SSTR2B. 20 These five SSTRs are widely distributed in various tissues, including the brain, pituitary, stomach, liver, pancreas, and intestine. 21,22 Therefore, SSTs show multiple physiological actions through SSTRs in a tissue-specific manner, such as the inhibition of GH and thyroid-stimulating hormone secretion from the anterior pituitary, inhibition of pancreatic endocrine and exocrine secretions, and inhibition and modulation of gastrointestinal functions. 23 SSTR5 is expressed in the pancreatic β-cells that secrete insulin 24,25 and intestinal L-cells that secrete gut hormones including glucagon-like peptide-1 (GLP-1) and peptide YY. 26 SST28 binds to SSTR5 with a 10-fold affinity higher than SST14 27 and is secreted after meals in humans 28 ; consequently, SSTR5 negatively regulates postprandial glucose excursion by inhibiting the secretion of these hormones. Therefore, several SSTR5 antagonists have been developed in the recent decade, and their therapeutic effects on glucose intolerance have been elucidated in animal models. [29][30][31][32][33][34] Interestingly, SSTR5 deletion has been reported to increase insulin sensitivity; however, the associated mechanisms have not been fully characterized. 35 In this study, we investigate the effects of SSTR5 inhibition on insulin resistance in vivo. First, we analyze the phenotype of high-fat diet (HFD)-fed SSTR5 knockout (KO) mice and evaluate the therapeutic potential of SSTR5 antagonism for diabetic phenotypes, including insulin resistance, using a novel selective SSTR5 antagonist (compound-1) 36 in male KK-A y /Ta Jcl mice (KK-A y mice), a model of obese type 2 diabetes with severe insulin resistance. Finally, we investigate the target organ by which selective SSTR5 antagonists alleviate insulin resistance using a hyperinsulinemic-euglycemic clamp technique in male KK-A y mice and measuring insulin-induced Akt phosphorylation in male C57BL/6J mice. The findings of this study provide deeper insights into the mechanism by which SSTR5 inhibition affects type 2 diabetes and will help to facilitate the development of effective therapeutic strategies against insulin resistance in type 2 diabetes.

| Materials
Compound-1 and its hydrochloride salt were synthesized by Takeda Pharmaceutical Company Limited. The chemical structure of compound-1 is shown in Figure 1

| Selectivity analysis of the SSTR subtypes
The activity of compound-1 on human SSTR subtypes other than SSTR5 was evaluated at a concentration of 10 μmol/L using a radioligand binding assay (Eurofins Panlabs Taiwan, Ltd.).

| Animals
The animal experiments were approved by the Institutional Animal

| HFD-fed SSTR5 KO study
To evaluate the metabolic parameters under the HFD feeding condition, male SSTR5 KO and wild-type (WT) littermate mice were fed a CE-2 diet or a HFD for 9-10 weeks from 8 weeks of age (CE-2; n = 8/ group, HFD; n = 22/group). At 17-18 weeks of age, body weight (BW) and food intake (FI) were measured, and blood samples were collected from the superficial temporal vein for the measurement of plasma parameters. Regarding FI data, one sample from HFD-fed WT mice and 2 samples from HFD-fed SSTR5 KO mice were lost because of a technical error. For the evaluation of insulin sensitivity, SSTR5 KO and WT mice were fed an HFD for 4 weeks from 14 weeks of age (n = 40/group). At 18 weeks, blood samples were collected from the tail vein after 6 h of fasting for plasma glucose (PG) and plasma insulin measurement.

| Repeated dose study of compound-1 in KK-A y mice
Male KK-A y mice aged 7 weeks were used for the dosage studies.
Animals were divided into four groups (n = 8/group) based on glycosylated hemoglobin (GHb), PG, insulin, and triglyceride (TG) levels; BW; FI; and change in BW during habituation. Mice were orally administered vehicle (0.5% MC) or compound-1 at a 1, 3, and 10 mg/ kg dosage once daily for 2 weeks (days 0-13). BW and FI were monitored every 1-3 days. On day 14, blood samples were collected from the tail vein of mice under fed or 6 h fasting conditions for the measurement of plasma parameters. Finally, the lean and fat mass were measured using the EchoMRI system (Aloka).

| Hyperinsulinemic-euglycemic clamp study in male KK-A y mice
At 7 weeks of age, male KK-A y mice were divided into two groups (vehicle group, n = 13; compound-1 group, n = 11) based on their glucose disappearance rate (Rd), and hepatic glucose production (HGP) were calculated using a modified formula based on a previous study. 37   The pS473 Akt and total Akt levels were measured using a commercially available kit (KHO0111 or KHO0101; Thermo Fisher Scientific) according to the manufacturer's instructions. One epididymal fat sample of "insulin + octreotide + compound−1"-treated mice was lost because of an operational error. Further, if the fasting insulin level was lower than the detection limit (20.28 μU/ml), as seen in three and five mice in the WT and KO groups, respectively (WT; n = 3/40, KO; n = 5/40), HOMA-IR was calculated using the following formula:

| Statistical analysis
All data represent the mean ± SD. To evaluate the effects of SSTR5 deletion in vivo, statistical differences between HFD-fed WT mice and HFD-fed SSTR5 KO mice were assessed using the Student's t-test and Aspin-Welch test. Conversely, to evaluate the effects of compound-1 in KK-A y mice, the statistical differences between the vehicle and drug treatments were assessed using the Student's ttest, Aspin-Welch test, and two-tailed Williams' test. In the insulininduced Akt phosphorylation study, Student's t-test or Aspin-Welch test was used to assess the comparisons between the control and insulin groups, insulin and insulin + octreotide groups, and between the insulin + octreotide and insulin + octreotide + compound-1 groups. A closed procedure was used to maintain the overall type-I error rate at 0.05. That is, the comparison between the control and insulin groups was assessed. If the resulting two-sided p-value was <0.05, the comparison between the insulin and insulin + octreotide groups was assessed. Next, if the two-sided p-value was <.05, the comparison between the insulin + octreotide and insulin + octreotide + compound-1 groups was assessed. For all tests, p < .05 was considered statistically significant. Statistical analysis was performed using SAS systems version 8.2 (SAS Institute Inc.) or EXSUS version 8.0 (CAC Croit Corporation).

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries. in http://www.guide topha rmaco logy. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 39

and are permanently archived in the Concise
Guide to PHARMACOLOGY 2019/20. 40

| Deletion of SSTR5 suppresses insulin resistance induced by HFD feeding
We investigated the effects of SSTR5 deletion on metabolic parameters under HFD feeding conditions in mice. Consistent with a previous report, 35 no significant difference was observed in HFD FI between WT and SSTR5 KO mice(WT = 2.96 g/day, KO = 2.82 g/day).
Although a HFD induced obesity and abnormal glucose metabolism, HFD-fed SSTR5 KO mice showed significantly decreased PG, plasma insulin, and GHb levels compared with HFD-fed WT mice ( Table 1). fasting insulin ( U ∕ ml) × fasting glucose (mg ∕ dl) ∕ 405 fasting insulin (20.28 U ∕ ml) × fasting glucose (mg ∕ dl) ∕ 405 To evaluate the effects of SSTR5 deletion on insulin resistance induced by HFD feeding for 4 weeks, we measured HOMA-IR, an index of insulin resistance. HOMA-IR was significantly decreased in HFD-fed SSTR5 KO mice compared to HFD-fed WT mice ( Figure 2).
These results suggest that the deletion of SSTR5 could maintain glucose homeostasis by improving insulin sensitivity.

| In vitro profiles of compound-1, a selective SSTR5 antagonist
The inhibitory activities of compound-1 against recombinant human and mouse SSTR5 were evaluated. The IC 50 values of compound-1 for human and mouse SSTR5 were 9.8 and 31 nmol/L, respectively ( Table 2).

| SSTR5 antagonist exhibits antidiabetic effects in KK-A y mice
To evaluate the therapeutic potential of SSTR5 antagonism for diabetic phenotypes, including insulin resistance, compound-1 was orally administered to KK-A y mice at 1, 3, and 10 mg/kg once daily for 2 weeks. In the vehicle-treated group, the GHb levels increased by 1.24% (from 4.50 ± 0.20% to 5.74 ± 0.53%) after 2 weeks of treatment ( Figure 3A); while in the compound-1-treated groups (1, 3, and 10 mg/kg), GHb levels increased by 0.88, 0.88, and 0.70%, respectively, after treatment ( Figure 3A). In particular, the increase in the 10 mg/kg compound 1-treated group was 0.54% less than that in the vehicle-treated group, suggesting the amelioration of the GHb level elevation by compound-1. Consistent with the reduction in GHb levels, compound-1 significantly decreased PG, plasma insulin, and HOMA-IR levels in a dose-dependent manner ( Figure 3B-D). No significant difference was observed in cumulative FI ( Figure 3E). BW was slightly increased in the compound-1-treated groups ( Figure 3F).
Measured using an EchoMRI system, the lean mass was significantly increased at ≧1 mg/kg and the fat mass was significantly increased at 10 mg/kg dose (data not shown). These results suggest that the selective SSTR5 antagonist improves insulin sensitivity independent of FI reduction or weight loss in an obese and diabetic animal model.

| SSTR5 antagonist improves hepatic insulin sensitivity in KK-A y mice
The hyperinsulinemic-euglycemic clamp study was performed in KK-A y mice under conscious and unrestrained conditions. KK-A y TA B L E 1 Effects of SSTR5 deletion on BW and plasma parameters under HFD feeding conditions. however, it did not alter Rd in KK-A y mice. These results suggest that the selective SSTR5 antagonist improves hepatic insulin sensitivity.

| SSTR5 antagonist alleviates the suppression effect of octreotide on insulin-induced Akt phosphorylation in the liver of C57BL/6J mice
To investigate the physiological role of SSTR5 on insulin signals, we evaluated the effect of octreotide, an SST analog that binds to SSTR2 and 5 with high affinity, 41  times, respectively, compared to the control group ( Figure 5). In the liver, octreotide inhibited insulin-induced Akt phosphorylation; this effect may have been negated by compound-1 ( Figure 5A). In con-

trast, neither octreotide nor compound-1 affected insulin-induced
Akt phosphorylation in other tissues ( Figure 5B-D). These results suggest that SST may attenuate insulin signaling in the liver in a tissue-specific manner, possibly via SSTR5 in mice.

| DISCUSS ION
SSTR5 is expressed in the pancreatic β-cells and intestinal L-cells; hence, previous studies have suggested that SSTR5 antagonism improves glucose tolerance via insulin and GLP-1 secretion. Additionally, SSTR5 might regulate insulin sensitivity; however, this has not been elucidated. 35 Therefore, the current study was performed to determine whether SSTR5 regulates insulin sensitivity in vivo using SSTR5 knockout mice and the selective SSTR5 antagonist.
Obesity is a critical factor for insulin resistance 42 ; hence, we in- Compound-1 significantly increased GIR and decreased HGP, indicating that an SSTR5 antagonist could increase hepatic insulin sensitivity. In addition, compound-1 abolished the inhibitory effect of octreotide on insulin-induced Akt phosphorylation in the liver of C57BL/6J mice. Moreover, the expression of hepatic G6pc, involved in HGP, was significantly reduced in HFD-fed SSTR5 KO mice compared with HFD-fed WT mice under fasting conditions in our preliminary studies ( Figure S3). These data support the findings of the clamp study that the selective SSTR5 antagonist inhibits hepatic insulin sensitivity.
Previous studies have reported that SSTR5 can potentially stimulate phosphotyrosine phosphatases (PTP) activity. 45,46 In addition, SSTRs, to which octreotide binds, have the potential to dephosphorylate the p85 subunit of phosphatidyl inositol 3 kinase (PI3K) via SHP-1, a classical PTP activation mechanism, resulting in inhibition of the insulin signal via suppression of PI3K/Akt activity. 47,48 Considering that both SSTR5 and insulin receptors are expressed in the liver, 22 an SSTR5 antagonist could rescue the insulin receptor/PI3K/Akt signaling by inhibiting the SSTR5-stimulated PTP activity in the liver.
Contrastingly, SST and SST analogs decrease the splanchnic blood flow and portal pressure in vivo. [49][50][51][52] In particular, octreotide and other SST analogs are thought to be useful for the management of gastroesophageal varices and variceal hemorrhage. 53 The underlying mechanism for SST-induced vasoconstriction can be elucidated using the phospholipase C/IP 3 activity/Ca 2+ mobilization 54 and PLA2-dependent arachidonic acid production pathways. 51 Moreover, the former potency could be the strongest at SSTR5 compared to other SSTR subtypes. 54 SST28 is secreted after meals, 28 and SSTR5 is constitutively expressed in the vascular smooth muscle cells, 55,56 which indicates that SST28/SSTR5 activation causes reduced exposure to insulin and glucose uptake in the liver postprandially. Therefore, the improvement in hepatic insulin sensitivity by the SSTR5 antagonist may be due to the recovery of the SST/SSTR5mediated hepatic blood flow decrease through SSTR5 blocking.
A limitation of our study is that we demonstrated that the target organ of SSTR5 antagonists is the liver using a hyperinsulinemic-euglycemic F I G U R E 3 Effects of compound-1 on diabetic parameters, food intake (FI), and body weight (BW) in KK-A y mice. Changes in levels of diabetic parameters: (A) glycosylated hemoglobin (GHb), (B) plasma glucose, (C) plasma insulin, and (D) homeostasis model assessment of insulin resistance (HOMA-IR); (E) cumulative FI, and (F) final BW are shown. Unshaded bar: before drug treatment; shaded bar: after drug treatment (B and C). Data represent the mean ± S.D. (n = 8). # p < .05, ## p < .01, and ### p < .001 compared to the vehicle group using the Williams' test.
clamp study and an insulin-induced Akt phosphorylation model utilizing only one insulin concentration in each of these. However, because each target organ has different sensitivities to the same insulin concentration, 57 the SSTR5 antagonist causes an increase in insulin sensitivity by the same mechanism in other SSTR5-expressing tissues, such as skeletal muscles 22,27,58,59 ; this assumption could not be denied completely and further investigation is needed.
In conclusion, we demonstrated that SSTR5 antagonists exhibit a glucose-lowering effect and improve insulin sensitivity in diabetic animal models independent of weight loss. Our results also suggest that the liver is one of the target organs of this mechanism; therefore, selective SSTR5 antagonists may represent a novel and attractive therapeutic agent for type 2 diabetes.

AUTH O R CO NTR I B UTI O N S
Research design: Yumiko Okano Tamura, Jun Sugama, Shin-ichi Abe, Yuji Shimizu, Hideki Hirose, Masanori Watanabe. Experimental procedures: Yumiko Okano Tamura, Jun Sugama, Shin-ichi Abe, Yuji F I G U R E 4 Effects of 0.03% hydrochloride salt of compound-1 on insulin sensitivity in KK-A y mice (A) Glucose disappearance rate (Rd), (B) glucose infusion rate (GIR), and (C) hepatic glucose production (HGP) based on hyperinsulinemic-euglycemic clamp analysis are presented. Data represent the mean ± SD (n = 11-13). *p < .05, compared with the vehicle group using the Student's t-test. $ p < .05, compared with the vehicle group using the Aspin-Welch test.

F I G U R E 5
Effect of compound-1 (10 mg/kg) on insulin-induced Akt phosphorylation in the tissues of C57BL/6J mice. The ratio of p-Akt to total Akt in the (A) liver, (B) gastrocnemius muscle, (C) epididymal fat, and (D) subcutaneous fat are presented. Data represent the mean ± SD (n = 4-5). *p < .05 using the Student's t-test, $ p < .05 using the Aspin-Welch test. A closed procedure was used to keep the overall type-I error rate at 0.05 (see Statistics section for more details).